The anticipated growth of cell culture-based virus production will require new paradigms for rapid, high-throughput, harvest, purification, concentration and formulation of a variety of human or non-human viruses. The greatest challenge is expected to come in the area of virus-based therapies such as vaccines and viral vectors for gene therapy. In such cases, the therapy will need to be custom manufactured for each patient. Traditional methods for virus purification, by their nature, are prone to technician error, inefficiency or inconsistency leading to low yield and high levels of impurities. This becomes especially evident as more and more applications for purified viruses come to light. Patient-specific therapies require relatively small quantities of the therapeutic agent and such small quantities are impossible to produce efficiently with traditional large scale manufacturing techniques.
With the increased awareness and monitoring of outbreaks of novel viral pathogens, the need has arisen for more efficient, rapid and consistent viral purification methods. Conventional purification techniques typically involve manually concentrating aqueous mixtures containing virus via centrifugation or passing the mixture of virus and impurities through a size exclusion gel filtration column that separates the virus from the mixture based on size. When the virus sized particles flow through the column they can be recovered in a somewhat purified form.
Manual methods for purifying viruses have their drawbacks. For example, these methods can be labor intensive, time consuming, and are highly inefficient. Large scale manufacturing techniques typically use multiple columns which are manually packed with resin and sterilized prior to each purification run. The manual steps involved in these methods also include a high risk of contamination.
Moreover, conventional approaches and tools for viruses or virus-based products typically involve numerous manual manipulations that are subject to variations even when conducted by skilled technicians. When used at the scale needed to manufacture hundreds or thousands of different cells, cell lines and patient-specific cell based therapies, the variability, error or contamination rate may become unacceptable for commercial processes.
Small quantities of viruses and virus-based products are produced via a number of cell culture techniques. T-flasks, roller bottles, stirred bottles, or cell bags are manual methods using incubators or warm-rooms to provide environments for cell growth and viral propagation. These methods are very labor intensive, have inconsistent performance, are subject to mistakes, and difficult for large-scale production.
Another method for producing viral-based products is by embryonated chicken eggs, which requires inoculating the allantoic cavity of fertilized chicken eggs with the virus of interest, which are thereby parasitically grown and maintained. The eggs are sacrificed and the virus is collected. This method is also very labor intensive, difficult for large scale production, and objectionable because of the potential for egg-based allergic reactions and variable productivity.
Another method for producing cell-secreted products involves inoculating cells growing in a small stirred tank or bioreactor or bag-type chamber. The tank provides the environmental and metabolic needs and the viruses are allowed to accumulate. This method is costly in terms of facility support in order to accommodate a large number of unique cells and produces product at low concentration.
Another method for the production of cell-secreted products is to use a bioreactor (e.g., hollow fiber, ceramic matrix, fluidizer bed, etc.) in lieu of the stirred tank. This can bring facilities costs down and increases product concentration. The systems currently available are general purpose in nature and require considerable time from trained operators to setup, load, flush, inoculate, run, harvest, and unload. Each step typically requires manual documentation, which is labor intensive and subject to errors.
Cell culturing devices or cultureware for culturing cells in vitro are known. Hollow fiber perfusion bioreactors (HFBx) were first introduced in 1972 as a model system to study tumors growing at tissue-like densities. Since then, HFBx have been used in a variety of applications such as bioartificial organs, pharmacokinetics, cell therapy, toxicology, etc. In the mid 1980s, Biovest International (formerly Endotronics, Inc.) developed the first commercial scale HFBx system and ever since, the most common application for this technology has been the large scale production of mammalian cell-secreted proteins, predominantly monoclonal antibodies.
As disclosed in U.S. Pat. No. 4,804,628, the entirety of which is hereby incorporated by reference, a hollow fiber culture device includes a plurality of hollow fiber membranes. Medium containing oxygen, nutrients, and other chemical stimuli is transported through the lumen of the hollow fiber membranes or capillaries and diffuses through the walls thereof into an extracapillary (EC) space between the membranes and the shell of the cartridge containing the hollow fibers. The cells that are to be maintained collect in the EC space. Metabolic wastes are removed from the bioreactor. The cells or cell products can be harvested from the device.
Each unique cell or cell line must be cultured, with viruses harvested and purified separately. In order to accommodate a large number of unique cells or cell lines, a considerable number of instruments would be needed. If application of the virus or virus-based products for therapeutic purposes is intended, strict segregation of each cell production and purification process would be required. Consequently, compactness of the design and the amount of ancillary support resources required will become an important facilities issue. Moreover, the systems currently available are general purpose in nature and require considerable time from trained operators to setup, load, flush, inoculate, run, harvest and unload. Each step usually requires manual documentation.
Moreover, production tracking mandates generation of a batch record for each cell culture and purification run. Historically, this is done with a paper-based system and relies on the operator inputting the information. This is labor intensive and subject to errors.
Current purification techniques also involve cleaning and reuse of equipment, chromatography columns and filtration media. This requires Standard Operational Procedures (SOPs) to be written and the cleaning and reuse process to be validated. This is a time intensive activity.
There is a need to provide alternative methods of manufacturing vaccines that protect against diseases caused by viruses, particularly highly pathogenic viruses, such as influenza strains H5N1 and H1N1. Furthermore, there remains a need to provide methods of manufacturing large quantities of vaccines in a time period rapid enough to effectively prevent possible epidemics and/or pandemics. In addition to the conventional egg-based production methods, mammalian cell culture-based production systems, and approaches using avian cell lines, insect cells, and plant cells have been proposed.
Large scale production of live virus and recombinant viral proteins in mammalian cells usually involves growing adherent cells in standard tissue culture flasks or roller bottles and non-adherent cells in spinner cultures. These systems utilize low density cell seeding in an excess of medium, and harvesting the cells at the point of medium exhaustion. While well-understood, robust and convenient, classical batch-style two-dimensional (2-D) culture on non-porous supports or three-dimensional (3-D) suspension culture in other devices do not replicate in vivo conditions and may affect protein modifications. Thus, in addition to efficient replication of virus, the production system must possess characteristics which will provide faithful production of viral proteins, particularly antigenic glycoproteins such as influenza hemagglutinin.
Accordingly, there is a need for a system and method whereby viruses and/or virus-based products can be cultured and purified in a fully automated, rapid and sterile manner.